Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V ka... more Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substant...
We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb... more We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.
Background: The extracellular matrix plays an important role in tissue regeneration. We investiga... more Background: The extracellular matrix plays an important role in tissue regeneration. We investigated whether extracellular matrix protein fragments could be targeted with antibodies to ischemically injured myocardium to promote angiogenesis and myocardial repair. Methodology/Principal Findings: Four peptides, 2 derived from fibronectin and 2 derived from Type IV Collagen, were assessed for in vitro and in vivo tendencies for angiogenesis. Three of the four peptides-Hep I, Hep III, RGD-were identified and shown to increase endothelial cell attachment, proliferation, migration and cell activation in vitro. By chemically conjugating these peptides to an anti-myosin heavy chain antibody, the peptides could be administered intravenously and specifically targeted to the site of the myocardial infarction. When administered into Sprague-Dawley rats that underwent ischemia-reperfusion myocardial infarction, these peptides produced statistically significantly higher levels of angiogenesis and arteriogenesis 6 weeks post treatment. Conclusions/Significance: We demonstrated that antibody-targeted ECM-derived peptides alone can be used to sufficiently alter the extracellular matrix microenvironment to induce a dramatic angiogenic response in the myocardial infarct area. Our results indicate a potentially new non-invasive strategy for repairing damaged tissue, as well as a novel tool for investigating in vivo cell biology.
A general method for directly obtaining the DNA sequence of the variable regions of any immunoglo... more A general method for directly obtaining the DNA sequence of the variable regions of any immunoglobulin chain by using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide or framework 1 and a conserved 3' constant region primer were used for enzymatic amplification of the heavy and light chain variable regions from human hybridomas producing monoclonal antibodies that recognize epitopes on HIV-1 (gp120, gp42), digoxin, tetanus, immunoglobulins (rheumatoid factor), and MHC class I and II proteins. Both ~, and /~ heavy chains and K and X light chains were successfully amplified and subsequently cloned. A similar strategy was used to amplify variable regions of mouse heavy and light chain variable regions from hydridomas recognizing human T cell antigens (CD3, CD6), an outer membrane protein of Neisseria meningiditis, carcinoembryonic antigen, fibrin, and PRP,the major carbohydrate antigen of Haemophilus influenza. Variable regions
It is hypothesized that methyl xanthines consumed in the strong tea brewed by Tibetan natives blu... more It is hypothesized that methyl xanthines consumed in the strong tea brewed by Tibetan natives blunt many of the effects of high altitude hypoxia.
Reye's syndrome affects children with a history of viral infection treated with aspirin. Its path... more Reye's syndrome affects children with a history of viral infection treated with aspirin. Its pathogenesis is unclear. Tumour necrosis factor (TNF) is released by macrophages activated by viral infection, endotoxin, and phagocytosis, and it has been shown to be a mediator of the toxic and metabolic effects of endotoxaemia. The metabolic effects of endotoxin and TNF are similar to those found in Reye's syndrome. Raised levels of TNF are released from macrophages treated with non-steroidal anti-inflammatory drugs, and young animals are known to be more sensitive than mature animals to both TNF and endotoxin. These observations lead to the hypothesis that an increased release of TNF in selected young patients treated with aspirin contributes to the development of Reye's syndrome.
Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that ma... more Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.
We evaluated the incidence of allergic and vasomotor symptoms, serum IgE concentrations, and the ... more We evaluated the incidence of allergic and vasomotor symptoms, serum IgE concentrations, and the cutaneous responses to allergens and/or methacholine in 229 Waorani Indians residing at 300 m altitude near the headwaters of the Amazon River, 39 Tibetans residing at 4000 m in the Himalayas, and 84 healthy subjects residing at 150 m in the piedmont region of North Carolina. The Waorani Indians had a high level of intestinal parasitism, an intermediate level of parasitism occurs in Tibetans, and parasitism is rare in the control population. One Waorani Indian (less than 1%), six Tibetans (15%), and 59 North Carolina subjects (88%) had a past history of allergic or vasomotor symptoms. The prevalence of positive epicutaneous allergen skin tests among the Waorani was 40 in 2910 tests and was significantly less (chi-squared = 184.5; p less than or equal to 0.0001) than the 151 in 1344 incidence in the North Carolina subjects. Large highly significant differences (p less than or equal to 0.0001) were detected between the geometric mean IgE concentrations (international unit per milliliter) and methacholine-induced cutaneous flare responsiveness (millimeter) elicited, respectively, in comparisons between the Waorani Indians (9806 IU/ml; less than 1.0 mm), Tibetans (2930 IU/ml; 2.06 mm), and North Carolina subjects (108 IU/ml; 4.49 mm). Differences in methacholine sensitivity were small and not significant. A highly significant inverse relationship (r = -0.50, p less than or equal to 0.0001) was detected between the circulating IgE concentrations and the methacholine-induced cutaneous flare responsiveness in this cross-cultural, cross-environmental comparison of three populations.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyperimmunoglobulinemia-E 185 Examination of these subjects revealed no evidence of bronchial con... more Hyperimmunoglobulinemia-E 185 Examination of these subjects revealed no evidence of bronchial constriction or dermatitis. Intestinal parasites were prevalent in the Waorani. Examination of single stool specimens in 65 of the subjects studied" revealed a 5 1% frequency of Entarnorhct co/i cysts, a 46% frequency of hook worm eggs, a 28% frequency of Gitrrtlitr Icrrnblia cysts, and a 207r frequency of E. hLro/yfic,c~ cysts. The frequency of A.scuris, Enterohius, and '/'rrch~tri.s
Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the d... more Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the development of immunodeficient mice (McCune et al. 1988) for the reconstitution of the human immune system ex vivo, immortalization of antigen-specific human B cells remains the limiting step in the generation of human monoclonal antibodies. Typically this is performed with the aid of Epstein-Barr virus transformation followed by subcloning, confirmation of antigen binding and hybridization of the B lymphoblasts to a suitable fusion partner such as GLI-H7. This general approach is effective and widely used; however, it is time-consuming with erratic results. These were the immediate reasons we and others devised methods to directly obtain the variable regions from small numbers of human B cells (Larrick et al. 1987). The success of the PCR-based approach is illustrated above. In the present studies we successfully captured and stably produced antibodies from the V regions of two potent human anti-tetanus antibodies secreted by heteromyelomas that were too unstable for scale-up production. Although further preclinical evaluation of these antibodies is in progress, results to date indicate that the recombinant antibodies produced in myeloma-based cell lines or CHO cells are equivalent in binding specificity and activity to the native heteromyeloma-derived antibodies. Recent studies from this laboratory indicate that effective anti-tetanus protection will require a cocktail of anti-tetanus antibodies. Details of this work will be the subject of a future communication (Lang et al., in preparation).
We have employed the circular dichroism (CD) technique to characterize the solution structure of ... more We have employed the circular dichroism (CD) technique to characterize the solution structure of CAP18~o6_~37, a lipopolysaccharide (LPS) binding, antimicrobial protein, and its interaction with lipid A. Our results revealed that CAP181o6_137 may exist in at least three lipid A concentration-dependent, primarily helix conformations. The 'model' structure of CAP181o6_~37 in 30% (v/v) TFE, determined by nuclear magnetic resonance (NMR) technique, was found to be a complete and very rigid helix. In this conformation, the cationic and hydrophobic groups of CAP18~o6_137 are separated into patches and stripes in such a way that it can favorably interact with lipid A through either coulombic interaction with the diphosphoryl groups or hydrophobic interaction with the fatty acyl chains.
Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V ka... more Using the polymerase chain reaction we examined for specific Ig kappa-L chain V region gene (V kappa gene) rearrangement in small lymphocytic non-Hodgkin's lymphomas that express Ig bearing a major kappa-L chain associated cross-reactive Id, designated 17.109. Previously, we identified the 17.109-cross-reactive Id in chronic lymphocytic leukemia as a serologic marker for expression of a highly conserved V kappa gene, designated Humkv325. Using sense-strand oligonucleotides specific for the 5'-end of this V kappa gene and antisense oligonucleotide specific for a J kappa region consensus sequence, we could amplify specifically Humkv325 when juxtaposed with J kappa through Ig gene rearrangement. This allowed us to amplify rearranged V kappa genes from DNA isolated from minute amounts of lymphoma biopsy material for molecular analyses. Our studies demonstrate that 17.109-reactive SL NHL, with or without associated CLL, rearrange, and presumably express, Humkv325 without substant...
We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb... more We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.
Background: The extracellular matrix plays an important role in tissue regeneration. We investiga... more Background: The extracellular matrix plays an important role in tissue regeneration. We investigated whether extracellular matrix protein fragments could be targeted with antibodies to ischemically injured myocardium to promote angiogenesis and myocardial repair. Methodology/Principal Findings: Four peptides, 2 derived from fibronectin and 2 derived from Type IV Collagen, were assessed for in vitro and in vivo tendencies for angiogenesis. Three of the four peptides-Hep I, Hep III, RGD-were identified and shown to increase endothelial cell attachment, proliferation, migration and cell activation in vitro. By chemically conjugating these peptides to an anti-myosin heavy chain antibody, the peptides could be administered intravenously and specifically targeted to the site of the myocardial infarction. When administered into Sprague-Dawley rats that underwent ischemia-reperfusion myocardial infarction, these peptides produced statistically significantly higher levels of angiogenesis and arteriogenesis 6 weeks post treatment. Conclusions/Significance: We demonstrated that antibody-targeted ECM-derived peptides alone can be used to sufficiently alter the extracellular matrix microenvironment to induce a dramatic angiogenic response in the myocardial infarct area. Our results indicate a potentially new non-invasive strategy for repairing damaged tissue, as well as a novel tool for investigating in vivo cell biology.
A general method for directly obtaining the DNA sequence of the variable regions of any immunoglo... more A general method for directly obtaining the DNA sequence of the variable regions of any immunoglobulin chain by using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide or framework 1 and a conserved 3' constant region primer were used for enzymatic amplification of the heavy and light chain variable regions from human hybridomas producing monoclonal antibodies that recognize epitopes on HIV-1 (gp120, gp42), digoxin, tetanus, immunoglobulins (rheumatoid factor), and MHC class I and II proteins. Both ~, and /~ heavy chains and K and X light chains were successfully amplified and subsequently cloned. A similar strategy was used to amplify variable regions of mouse heavy and light chain variable regions from hydridomas recognizing human T cell antigens (CD3, CD6), an outer membrane protein of Neisseria meningiditis, carcinoembryonic antigen, fibrin, and PRP,the major carbohydrate antigen of Haemophilus influenza. Variable regions
It is hypothesized that methyl xanthines consumed in the strong tea brewed by Tibetan natives blu... more It is hypothesized that methyl xanthines consumed in the strong tea brewed by Tibetan natives blunt many of the effects of high altitude hypoxia.
Reye's syndrome affects children with a history of viral infection treated with aspirin. Its path... more Reye's syndrome affects children with a history of viral infection treated with aspirin. Its pathogenesis is unclear. Tumour necrosis factor (TNF) is released by macrophages activated by viral infection, endotoxin, and phagocytosis, and it has been shown to be a mediator of the toxic and metabolic effects of endotoxaemia. The metabolic effects of endotoxin and TNF are similar to those found in Reye's syndrome. Raised levels of TNF are released from macrophages treated with non-steroidal anti-inflammatory drugs, and young animals are known to be more sensitive than mature animals to both TNF and endotoxin. These observations lead to the hypothesis that an increased release of TNF in selected young patients treated with aspirin contributes to the development of Reye's syndrome.
Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that ma... more Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.
We evaluated the incidence of allergic and vasomotor symptoms, serum IgE concentrations, and the ... more We evaluated the incidence of allergic and vasomotor symptoms, serum IgE concentrations, and the cutaneous responses to allergens and/or methacholine in 229 Waorani Indians residing at 300 m altitude near the headwaters of the Amazon River, 39 Tibetans residing at 4000 m in the Himalayas, and 84 healthy subjects residing at 150 m in the piedmont region of North Carolina. The Waorani Indians had a high level of intestinal parasitism, an intermediate level of parasitism occurs in Tibetans, and parasitism is rare in the control population. One Waorani Indian (less than 1%), six Tibetans (15%), and 59 North Carolina subjects (88%) had a past history of allergic or vasomotor symptoms. The prevalence of positive epicutaneous allergen skin tests among the Waorani was 40 in 2910 tests and was significantly less (chi-squared = 184.5; p less than or equal to 0.0001) than the 151 in 1344 incidence in the North Carolina subjects. Large highly significant differences (p less than or equal to 0.0001) were detected between the geometric mean IgE concentrations (international unit per milliliter) and methacholine-induced cutaneous flare responsiveness (millimeter) elicited, respectively, in comparisons between the Waorani Indians (9806 IU/ml; less than 1.0 mm), Tibetans (2930 IU/ml; 2.06 mm), and North Carolina subjects (108 IU/ml; 4.49 mm). Differences in methacholine sensitivity were small and not significant. A highly significant inverse relationship (r = -0.50, p less than or equal to 0.0001) was detected between the circulating IgE concentrations and the methacholine-induced cutaneous flare responsiveness in this cross-cultural, cross-environmental comparison of three populations.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyperimmunoglobulinemia-E 185 Examination of these subjects revealed no evidence of bronchial con... more Hyperimmunoglobulinemia-E 185 Examination of these subjects revealed no evidence of bronchial constriction or dermatitis. Intestinal parasites were prevalent in the Waorani. Examination of single stool specimens in 65 of the subjects studied" revealed a 5 1% frequency of Entarnorhct co/i cysts, a 46% frequency of hook worm eggs, a 28% frequency of Gitrrtlitr Icrrnblia cysts, and a 207r frequency of E. hLro/yfic,c~ cysts. The frequency of A.scuris, Enterohius, and '/'rrch~tri.s
Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the d... more Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the development of immunodeficient mice (McCune et al. 1988) for the reconstitution of the human immune system ex vivo, immortalization of antigen-specific human B cells remains the limiting step in the generation of human monoclonal antibodies. Typically this is performed with the aid of Epstein-Barr virus transformation followed by subcloning, confirmation of antigen binding and hybridization of the B lymphoblasts to a suitable fusion partner such as GLI-H7. This general approach is effective and widely used; however, it is time-consuming with erratic results. These were the immediate reasons we and others devised methods to directly obtain the variable regions from small numbers of human B cells (Larrick et al. 1987). The success of the PCR-based approach is illustrated above. In the present studies we successfully captured and stably produced antibodies from the V regions of two potent human anti-tetanus antibodies secreted by heteromyelomas that were too unstable for scale-up production. Although further preclinical evaluation of these antibodies is in progress, results to date indicate that the recombinant antibodies produced in myeloma-based cell lines or CHO cells are equivalent in binding specificity and activity to the native heteromyeloma-derived antibodies. Recent studies from this laboratory indicate that effective anti-tetanus protection will require a cocktail of anti-tetanus antibodies. Details of this work will be the subject of a future communication (Lang et al., in preparation).
We have employed the circular dichroism (CD) technique to characterize the solution structure of ... more We have employed the circular dichroism (CD) technique to characterize the solution structure of CAP18~o6_~37, a lipopolysaccharide (LPS) binding, antimicrobial protein, and its interaction with lipid A. Our results revealed that CAP181o6_137 may exist in at least three lipid A concentration-dependent, primarily helix conformations. The 'model' structure of CAP181o6_~37 in 30% (v/v) TFE, determined by nuclear magnetic resonance (NMR) technique, was found to be a complete and very rigid helix. In this conformation, the cationic and hydrophobic groups of CAP18~o6_137 are separated into patches and stripes in such a way that it can favorably interact with lipid A through either coulombic interaction with the diphosphoryl groups or hydrophobic interaction with the fatty acyl chains.
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Papers by J. Larrick